Armed Dual CAR-T Compositions and Methods For Cancer Immunotherapy

Abstract

The disclosure provides, in various embodiments, polynucleotides and vectors comprising sequences encoding a mono-specific or a bi-specific CAR that is capable of binding to a first TAA, or a T-cell engager that is capable of binding to CD3 and a second TAA, or a combination thereof. The disclosure also provides, in various embodiments, T lymphocytes comprising one or more of the polynucleotides or vectors; compositions (e.g., pharmaceutical compositions) and kits comprising one or more of the T lymphocytes; methods of treating a cancer in mammalian subject (e.g., a human), and methods of inducing T cell-mediated cytolysis of cancer cells (e.g., solid tumor cells).

1 . A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective dosage of T lymphocytes, wherein a plurality of the T lymphocytes comprise a polynucleotide encoding a fusion protein that comprises a chimeric antigen receptor (CAR) and a T-cell engager, wherein the fusion protein is expressed and subsequently cleaved in the T lymphocytes to produce a separated CAR and a separated T-cell engager, and wherein the separated CAR is expressed at the cell surface of the T lymphocytes and the separated T-cell engager is secreted by the T lymphocytes. 
     
     
         2 . The method of  claim 1 , wherein the CAR is capable of binding to one or more first tumor associated antigens (TAAs) and the T-cell engager is capable of binding to a T-cell and a second TAA. 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 2 , wherein the CAR is capable of binding to two epitopes of a first TAA. 
     
     
         6 . The method of  claim 2 , wherein the one or more first TAAs each is independently selected from interleukin-13 receptor subunit alpha-2 (IL13Rα2), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), glypican-3 (GPC3), or any combination thereof. 
     
     
         7 . The method of  claim 1 , wherein the CAR comprises a mutein, a single-chain variable fragment (scFv), a nanobody, or a combination thereof. 
     
     
         8 . The method of  claim 1 , wherein the T-cell engager is capable of binding to CD3. 
     
     
         9 . The method of  claim 1 , wherein the fusion protein comprises an amino acid sequence having at least 90% sequence identity to at least one amino acid sequence set forth in SEQ ID NOs: 35-38; optionally, wherein the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 35-38. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the cancer is a hematologic cancer or a solid tumor; optionally, wherein the solid tumor is a brain tumor, a breast cancer, a lung cancer, or a liver cancer. 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 15 , wherein the brain tumor is glioblastoma (GBM), optionally, wherein the GBM is recurrent or primary glioblastoma multiforme; or wherein the brain tumor is a brain metastatic tumor, optionally, wherein the brain metastatic tumor is non-small cell lung cancer brain metastases (NSCLCBM), small cell lung cancer brain metastases (SCLCBM), HER2-positive metastatic breast cancer, or triple-negative breast cancer brain metastases (TNBCBM). 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 15 , wherein the liver cancer is hepatocellular carcinoma (HCC). 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the subject is newly diagnosed with cancer or has relapsed from or is refractory to a prior cancer therapy. 
     
     
         23 . (canceled) 
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein at least 10% of the T lymphocytes express the fusion protein; optionally, wherein about 15-75% of the T lymphocytes express the fusion protein. 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 1 , wherein the subject received a chemotherapy before administration of the T lymphocytes. 
     
     
         32 . A method of inducing T cell-mediated cytolysis of cancer cells, comprising contacting the cancer cells with an effective dosage of T lymphocytes, wherein a plurality of the T lymphocytes comprise a polynucleotide encoding a fusion protein that comprises a chimeric antigen receptor (CAR) and a T-cell engager, wherein the fusion protein is expressed and subsequently cleaved in the T lymphocytes to produce a separated CAR and a separated T-cell engager, and wherein the separated CAR is expressed at the cell surface of the T lymphocytes and the separated T-cell engager is secreted by the T lymphocytes. 
     
     
         33 . The method of  claim 32 , wherein the cancer cells are cells of a hematologic cancer or a solid tumor; optionally, wherein the solid tumor is a brain tumor, a breast cancer tumor, a lung cancer tumor, or a liver cancer tumor. 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . The method of  claim 33 , wherein the brain tumor is glioblastoma (GBM), optionally, wherein the GBM is recurrent or primary glioblastoma multiforme; or the brain tumor is a brain metastatic tumor, optionally, wherein the brain metastatic tumor is non-small cell lung cancer brain metastases (NSCLCBM), small cell lung cancer brain metastases (SCLCBM), HER2-positive metastatic breast cancer, or triple-negative breast cancer brain metastases (TNBCBM). 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . The method of  claim 33 , wherein the liver cancer is hepatocellular carcinoma (HCC). 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . (canceled) 
     
     
         46 . The method of  claim 32 , wherein the CAR comprises a mutein, a single-chain variable fragment (scFv), a nanobody, or a combination thereof. 
     
     
         47 . The method of  claim 32 , wherein the T-cell engager is capable of binding to CD3. 
     
     
         48 . The method of  claim 32 , wherein the fusion protein comprises an amino acid sequence having at least 90% sequence identity to at least one amino acid sequence set forth in SEQ ID NOs: 35-38; optionally, wherein the fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 35-38. 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . (canceled) 
     
     
         52 . (canceled) 
     
     
         53 . (canceled)